The goal of these studies is to characterize the large and complex virion of Salmonella phage SPN3US and to establish a genetic system to facilitate studies of the fundamental features of giant phages and how they parasitize their hosts. This research is significant as recent studies have shown that giant phages abound in the environment. However, the long genomes of giant phages (>200 kb) encode many genes of which typically 80% are functionally uncharacterized. That is, essentially we have extremely limited understanding as to the molecular mechanisms of giant phage infection within the host cell. This is a problem because most giant phages were isolated with the goal of using them for phage therapy to treat multi- drug resistant pathogens or other novel biocontrol applications. The lack of knowledge regarding giant phages represents a significant hurdle to obtaining regulatory approval for therapeutic phages. Additionally, without insight into the processes of host takeover and infection, there can be no rational optimization of the most appropriate phage(s) to select for therapeutics or identification of phage proteins to target for further research as novel biotechnological tools. This research will address the lack of fundamental knowledge about giant phages by the characterization of Salmonella phage SPN3US as a model for an expanding group of phages that infect human pathogens. Preliminary studies have shown SPN3US is a suitable model phage sharing a core set of genes with other giant phages. In addition, SPN3US infects the genetically tractable host Salmonella Typhimurium LT2 which has facilitated the isolation of SPN3US amber mutants, the first such collection for any giant phage. Our studies have shown SPN3US is an extraordinarily large tailed phage with novel structural features and is comprised of >70 different proteins. As with all tailed phages, the role of the SPN3US capsid or head is to protect the phage genome while in the environment to enable its delivery to a new bacterium. We hypothesize that the SPN3US head has evolved an additional role to transport a large cargo of ejection proteins that enter the Salmonella cell with the dsDNA and ensure its subordination to the goal of viral progeny production. Consequently, we also hypothesize that giant phages have a set of essential virion proteins to achieve both these roles. This research aims to test these hypotheses with the following three aims: (1) An in-depth characterization of the SPN3US virion and head to identify all component proteins and the copy numbers of each per phage particle. This will be achieved via structural analyses using Cryo- electron microscopy and proteomic analyses using mass spectrometry. (2) Identification of the essential proteins in the SPN3US virion. This aim will be achieved via the isolation and genome sequencing of amber mutant phages, and (3) Delineation and characterization of SPN3US head proteins that are ejection proteins via biochemical, structural and proteomic analyses of mutant phage particles.